Method for nucleic acid amplification

ABSTRACT

A kit for amplifying a nucleic acid, using RNA as a template, which can realize elimination of the risk of non-specific amplification caused by DNA mixed from reagents and/or working environment, an increase in the detection sensitivity of trace RNA, and a reduction in amplification bias. The kit includes a degrading enzyme specific to DNA in an RNA-DNA hybrid that is a double strand-specific DNA degrading enzyme or a non-specific DNA degrading enzyme, and an RNase H minus reverse transcriptase. If the degrading enzyme specific to DNA in an RNA-DNA hybrid is a non-specific DNA degrading enzyme, then the kit further comprises a single-stranded DNA-binding protein.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a Divisional of U.S. application Ser. No.15/514,931, which is a National Stage of PCT/JP2015/077745, filed Sep.30, 2015, which claims priority to JP App. No. 2014-200258, filed Sep.30, 2014. Each of the above applications is incorporated by referenceherein in its entirety.

TECHNICAL FIELD

The present invention relates to a method for amplifying a nucleic acid,in which a degrading enzyme specific to DNA in RNA-DNA hybrid and astrand displacement activity are used. More specifically, the presentinvention relates to a method for amplifying a nucleic acid, using RNAas a template, and also using a degrading enzyme specific to DNA inRNA-DNA hybrid and an RNase H minus reverse transcriptase. Moreover, thepresent invention relates to a kit used for performing theabove-described method for amplifying a nucleic acid.

BACKGROUND ART

A large number of methods for amplifying a nucleic acid have beenreported so far. Examples of such a method for amplifying a nucleic acidinclude Polymerase Chain Reaction (PCR) (Non Patent Literature 1),Strand Displacement Amplification (SDA) (Non Patent Literature 2),Multiple Displacement Amplification (MDA) (Non Patent Literature 3),Rolling-Circle Amplification (RCA) (Non Patent Literature 4),Loop-Mediated Isothermal Amplification (LAMP) (Non Patent Literature 5),Smart Amplification Process (SmartAmp) (Non Patent Literature 6),Helicase-Dependent Amplification (HDA) (Non Patent Literature 7), andLigase Chain Reaction (LCR) (Non Patent Literature 8). These methods areamplification methods of using DNA as a template, and in all of themethods other than PCR, HDA and LCR, the strand displacement activity ofDNA polymerase is utilized. A main amplification system of utilizing astrand displacement reaction is SDA (Non Patent Literature 2). In SDA, acleavage site (nick) is made on one strand of double-stranded DNA usinga restriction enzyme or the like. Using this nick as a starting point,the DNA strand on the 3′ side is peeled by the strand displacementactivity of DNA polymerase, and novel complementary DNA is synthesized.SDA is a technique of amplifying complementary DNA by continuouslygenerating this reaction. Moreover, MDA, which utilizes a random hexamerprimer, is able to randomly take place a strand displacement reaction byrandomly annealing primers to multiple sites on template DNA, and thus,this is a method having an extremely high amplification rate (Non PatentLiterature 3). However, in order to amplify an RNA sequence according tothese amplification methods using DNA as a template, it is necessary toperform a reverse transcription reaction of converting RNA to DNA. Inaddition, in the case of SDA, since a nick needs to be inserted intotemplate DNA using restriction enzymes, it is necessary to add arestriction enzyme recognition sequence, deoxyinosine and the like tothe template. Moreover, in the case of LAMP and SmartAmp, four or fivesequence-specific primers are necessary for a single target. Thus, thesynthesis of a special oligo primer, the designing of asequence-specific primer, and the like become necessary. Furthermore,amplification methods using DNA as a template, such as PCR as arepresentative example, are problematic in terms of pseudo-positiveresults generated as a result of the carry-over of the previous PCRamplification products or reverse transcription products on samples, andnon-specific products derived from DNA contaminated with reagents, DNAmixed from working environment, etc. (Non Patent Literatures 9 and 10).In particular, in the case of MDA, since primers are randomly annealedto DNA, it is likely that such contaminated DNA-derived non-specificproducts would be increased. As an amplification technique of using RNAas a template, a method of utilizing a special DNA hairpin primer havinga restriction enzyme recognition sequence has been proposed. In thismethod, a cleavage point is generated on a DNA hairpin primer that hasbeen allowed to bind to RNA as a template, and then, a stranddisplacement reaction is allowed to take place, so as to amplifycomplementary strand DNA. In this method, however, a pre-treatment needsto be performed to ligate a DNA hairpin primer to the 3′-end of templateRNA, and thus, molecules that cannot be captured are likely to begenerated depending on ligation conditions. Further, since amplificationis started from the 3′-end of template RNA, it is considered difficultfor this method to equally amplify the entire-length DNA.

PRIOR ART LITERATURES Non Patent Literatures

Non Patent Literature 1: Specific Enzymatic Amplification of DNA InVitro: The Polymerase Chain Reaction. Mullis Ket, al., Cold Spring HarbSymp Quant Biol. 1986; 51 Pt 1: 263-73.

Non Patent Literature 2: Isothermal in vitro amplification of DNA by arestriction enzyme/DNA polymerase system. Walker G T, et al., Proc NatlAcad Sci USA. 1992 Jan. 1; 89(1): 392-6.

Non Patent Literature 3: Comprehensive human genome amplification usingmultiple displacement amplification. Dean F B, et al., Proc Natl AcadSci USA. 2002 Apr. 16; 99(8): 5261-6.

Non Patent Literature 4: Mutation detection and single-molecule countingusing isothermal rolling-circle amplification. Lizardi P M, et al., NatGenet. 1998 Jul.; 19(3): 225-32.

Non Patent Literature 5: Loop-mediated isothermal amplification of DNA.Notomi T, et al., Nucleic Acids Res. 2000 Jun. 15; 28(12): E63.

Non Patent Literature 6: Rapid SNP diagnostics using asymmetricisothermal amplification and a new mismatch-suppression technology.Mitani Y, et al., Nat Methods. 2007 Mar.; 4(3): 257-62. Epub 2007 Feb.18.

Non Patent Literature 7: Helicase-dependent isothermal DNAamplification. Myriam Vincent, et al., EMBO Rep. 2004 Aug.; 5(8):795-800.

Non Patent Literature 8: Genetic disease detection and DNA amplificationusing cloned thermostable ligase. Barany F. Proc Natl Acad Sci USA. 1991Jan. 1; 88(1): 189-93.

Non Patent Literature 9: An Efficient Multistrategy DNA DecontaminationProcedure of PCR Reagents for Hypersensitive PCR Applications. ChamplotS, et al., PLoS One. 2010 Sep. 28; 5(9).

Non Patent Literature 10: Novel Sensitive Real-Time PCR forQuantification of Bacterial 16S rRNA Genes in Plasma of HIV-InfectedPatients as a Marker for Microbial Translocation. Kramski M, et al., JClin Microbiol. 2011 October; 49(10)

Non Patent Literature 11: A Novel Method for SNP Detection Using a NewDuplex-Specific Nuclease From Crab Hepatopancreas. Shagin D A, et al.,Genome Res. 2002 Dec.; 12(12): 1935-42

Non Patent Literature 12: The Enzyme and the cDNA Sequence of aThermolabile and Double-Strand Specific DNase from Northern Shrimps(Pandalus borealis). Nilsen I W, et al., PLoS One. 2010 Apr. 22; 5(4):e10295.

Non Patent Literature 13: Experimental Murine Endometriosis Induces DNAMethylation and Altered Gene Expression in Eutopic Endometrium 1. Lee B,et al., Biol Reprod. 2009 January; 80(1): 79-85.

Non Patent Literature 14: Versatile synthesis ofoligodeoxyribonucleotide-oligospermine conjugates. Voirin E, Behr J P,et al., Nat Protoc. 2007; 2(6): 1360-7.

Non Patent Literature 15: Oligonucleotide-oligospermine conjugates (zipnucleic acids): a convenient means of finely tuning hybridizationtemperatures. Noir R, et al., J Am Chem Soc. 2008 Oct. 8; 130(40):13500-5.

Non Patent Literature 16: Zip Nucleic Acids: new high affinityoligonucleotides as potent primers for PCR and reverse transcription.Moreau V, et al., Nucleic Acids Res. 2009 October; 37(19)

Non Patent Literature 17: DNA “melting” proteins. IV. Fluorescencemeasurements of binding parameters for bacteriophage T4 gene 32-proteinto mono-, oligo-, and polynucleotides. Kelly R C, et al., J Biol Chem.1976 Nov. 25; 251(22): 7240-50.

Non Patent Literature 18: Reverse Transcriptase (RT) Inhibition of PCRat Low Concentrations of Template and Its Implications for QuantitativeRT-PCR. Chandler D P, et al., Appl Environ Microbiol. 1998 February;64(2): 669-77.

Non Patent Literature 19: Increased Yield of PCR Products by Addition ofT4 Gene 32 Protein to the SMART-PCR cDNA Synthesis System. Villalva C,et al., Biotechniques. 2001 Jul.; 31(1): 81-3, 86.

Non Patent Literature 20: An Optimized Protocol for First Strand cDNASynthesis from Laser Capture Microdissected Tissue. Boylan S, et al.,Lab Invest. 2001 August; 81(8): 1167-9.

Non Patent Literature 21: Extra-embryonic endoderm cells derived from EScells induced by GATA factors acquire the character of XEN cells.Shimosato D, et al., BMC Dev Biol. 2007 Jul. 3; 7: 80.

Non Patent Literature 22: Characterization of SYBR Gold nucleic acid gelstain: a dye optimized for use with 300-nm ultraviolettransilluminators. Tuma R S, et al., Anal Biochem. 1999 Mar. 15; 268(2):278-88.

Non Patent Literature 23: CRYSTALLINE DESOXYRIBONUCLEASE II. DIGESTIONOF THYMUS NUCLEIC ACID (DESOXYRIBONUCLEIC ACID) THE KINETICS OF THEREACTION. Kunitz M, J Gen Physiol. 1950 March; 33(4): 363-77.

Non Patent Literature 24: The effect of divalent cations on the mode ofaction of DNase I. The initial reaction products produced fromcovalently closed circular DNA. Campbell V W, et al., J Biol Chem. 1980Apr. 25; 255(8): 3726-35.

Non Patent Literature 25: Crystal structure of a replication forksingle-stranded DNA binding protein (T4 gp32) complexed to DNA. ShamooY, et al., Nature. 1995 Jul. 27; 376(6538): 362-6.

SUMMARY OF INVENTION Object to be Solved by the Invention

To perform a gene expression analysis, it is necessary to convert RNA tocomplementary DNA (cDNA). There has been an amplification technique ofusing DNA as a template, but there have been no amplification techniquesof directly using, as a template, untreated RNA that has not beensubjected to a pre-treatment such as addition of a special sequence. Itis an object of the present invention to provide, not the existingamplification method of using DNA as a template, but a method foramplifying a nucleic acid, using RNA as a template. That is, it is anobject of the present invention to provide a method for amplifying anucleic acid, using RNA as a template, which can realize not onlysimplification of operations, but also elimination of the risk ofnon-specific amplification caused by DNA contaminated with reagentsand/or working environment, an increase in the detection sensitivity oftrace RNA, and a reduction in amplification bias.

Means for Solving the Object

As a result of intensive studies directed towards achieving theaforementioned object, the present inventors have discovered a reversetranscription method with random displacement amplification (hereinafteralso referred to as a “RT-RamDA method,” or simply a “RamDA method”), inwhich cDNA is amplified using RNA as a template. It have been revealedthat, according to the RT-RamDA method, the yield of cDNA can beincreased to 10 to 100 times as compared to a commercially availablekit, and that it becomes possible to capture low expressed genes or toincrease the number of detected genes in a gene expression analysis froma trace amount of RNA. Moreover, the cDNA amplified by the RamDA methodcan be applied to various gene analyses such as an RNA sequence method,and thus, it is expected that the RamDA method will be used as a basictechnique of molecular biology in a wide range of fields. Furthermore,differing from the existing amplification technology of using DNA as atemplate, the use of RNA as a template can realize not onlysimplification of operations, but also elimination of the risk ofnon-specific amplification caused by DNA contaminated with reagentsand/or working environment, and a reduction in amplification bias. Thepresent invention has been completed based on these findings.

Specifically, according to the present invention, the followinginventions are provided.

-   (1) A method for amplifying a nucleic acid, comprising a step of    incubating a mixture containing template RNA, a primer, a degrading    enzyme specific to DNA in RNA-DNA hybrid, an RNase H minus reverse    transcriptase, and a substrate, wherein the degrading enzyme    specific to DNA in RNA-DNA hybrid has an activity of cleaving a DNA    strand in the RNA-DNA hybrid.-   (2) The method for amplifying a nucleic acid according to (1), which    comprises a step of synthesizing a complementary strand DNA (cDNA)    of the template RNA by the RNA-dependent DNA polymerase activity of    the RNase H minus reverse transcriptase, then randomly cleaving the    cDNA strand in the hybrid strand of RNA and cDNA by the degrading    enzyme specific to DNA in RNA-DNA hybrid, then peeling the cDNA    strand on the 3′ side from the RNA by the strand displacement    activity of the RNase H minus reverse transcriptase, while using the    cleavage site as a starting point, and then synthesizing a novel    cDNA strand in a portion peeled by the RNase H minus reverse    transcriptase.-   (3) The method for amplifying a nucleic acid according to (1) or    (2), wherein the mixture comprises a double strand-specific DNA    degrading enzyme as a degrading enzyme specific to DNA in RNA-DNA    hybrid, and the double strand-specific DNA degrading enzyme has an    activity of cleaving the DNA strand in the RNA-DNA hybrid, and    substantially does not have an activity of cleaving the RNA strand    in the RNA-DNA hybrid, single-stranded DNA and single-stranded RNA.-   (4) The method for amplifying a nucleic acid according to (1) or    (2), wherein the mixture comprises a non-specific DNA degrading    enzyme as a degrading enzyme specific to DNA in RNA-DNA hybrid, and    the non-specific DNA degrading enzyme has an activity of cleaving    the DNA strand of the RNA-DNA hybrid strand, and substantially does    not have an activity of cleaving the RNA strand in the RNA-DNA    hybrid, and single-stranded RNA.-   (5) The method for amplifying a nucleic acid according to any one    of (1) to (4), wherein the degrading enzyme specific to DNA in    RNA-DNA hybrid has a DNA-degrading activity even at a temperature of    lower than 60° C.-   (6) The method for amplifying a nucleic acid according to (3),    wherein the double strand-specific DNA degrading enzyme is a double    strand-specific DNA degrading enzyme derived from Crustacea, or a    variant thereof.-   (7) The method for amplifying a nucleic acid according to (6),    wherein the double strand-specific DNA degrading enzyme is a double    strand-specific DNA degrading enzyme derived from a shrimp, or a    variant thereof.-   (8) The method for amplifying a nucleic acid according to (4),    wherein the non-specific DNA degrading enzyme is a non-specific DNA    degrading enzyme derived from a mammal, or a variant thereof.-   (9) The method for amplifying a nucleic acid according to (8),    wherein the non-specific DNA degrading enzyme is a non-specific DNA    degrading enzyme derived from a bovine, or a variant thereof.-   (10) The method for amplifying a nucleic acid according to any one    of (1) to (9), wherein the primer is one or more of a random primer,    an oligo dT primer and a sequence-specific primer.-   (11) The method for amplifying a nucleic acid according to any one    of (1) to (10), wherein the primer is a primer, the Tm value of    which is increased by modification with a cation unit.-   (12) The method for amplifying a nucleic acid according to any one    of (1) to (11), wherein the primer is a Zip Nucleic Acid (ZNA)    primer.-   (13) The method for amplifying a nucleic acid according to any one    of (1) to (12), wherein the mixture further comprises a    single-stranded DNA-binding protein.-   (14) The method for amplifying a nucleic acid according to any one    of (1) to (13), wherein the template RNA is trace RNA corresponding    to a volume ranging from a single cell to several hundreds of cells.-   (15) The method for amplifying a nucleic acid according to any one    of (1) to (14), which is performed to amplify cDNA that is to be    subjected to the production of a DNA sequence library.-   (16) A kit used for performing the method for amplifying a nucleic    acid according to any one of (1) to (15), wherein the kit comprises,    at least, a degrading enzyme specific to DNA in RNA-DNA hybrid and    an RNase H minus reverse transcriptase.-   (17) The kit according to (16), which further comprises a    single-stranded DNA-binding protein.

Advantageous Effects of Invention

Since a nucleic acid can be amplified using RNA as a template accordingto the method for amplifying a nucleic acid of the present invention,the present method can realize not only simplification of operations,but also elimination of the risk of non-specific amplification caused byDNA contaminated with reagents and/or working environment, and areduction in amplification bias. Moreover, according to the method foramplifying a nucleic acid of the present invention, the yield that is 10times or more the yield as compared to the existing reversetranscription cDNA synthesis kit can be obtained, and it is alsopossible to perform application at a magnification of 100 times or more,depending on conditions.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 is a schematic view of an RT-RamDA (RamDA) method.

FIG. 2 shows that a double strand-specific DNA degrading enzyme(dsDNase) and a non-specific DNA degrading enzyme (DNase I) cleave notonly double-stranded DNA, but also DNA in an RNA-DNA hybrid. Accordingto FRET analysis using a fluorescent oligonucleotide, dsDNase and DNaseI were examined in terms of the properties of nuclease under reservetranscription buffer conditions. The nuclease activity was measured overtime under four conditions, namely, dsDNase, DNase I, HL-dsDNase havingthermal instability, and a buffer control (buffer) that did not containDNase. FIGS. 2A and 2C show the activity in DNA of double-stranded DNA,FIG. 2B shows the activity in DNA of an RNA-DNA hybrid, and FIG. 2Dshows the activity in RNA of the hybrid. FIGS. 2E and 2F show theresults of the activity in single-stranded DNA and in single-strandedRNA, respectively. The measurement was carried out by performingdetection 50 times, at 37° C. every 70 seconds. The error bar indicatesa standard deviation (n=3).

FIG. 3 shows that fragmentation and amplification of cDNA take place bythe activity of dsDNase. In FIG. 3A, using 5 ng of artificiallysynthesized RNA, Thr RNA (2,052 b), as a template, the cDNA pattern of astandard reverse transcription method (RamDA (−)) and the cDNA patternof RamDA (+) involving addition of dsDNase, a ZNA-random hexamer primerand a T4 gene 32 protein to the standard reverse transcription methodwere examined by agarose gel electrophoresis. The ssDNA ladder is athermally denatured dsDNA ladder. The arrows each indicate 2 kb. In thefigure, the symbol “−” indicates RamDA (−), and the symbol “+” indicatesRamDA (+). In FIG. 3B, the amount of cDNA in 100 fg of artificiallysynthesized RNAs, Dap RNA (96,725 copies) and Thr RNA (89,991 copies),which were added to 10 pg of total RNA derived from mouse ES cells thatwas the amount of total RNA corresponding to a single cell, was detectedby qPCR. For the qPCR, a reverse transcription product was used in anamount of 1/50. The horizontal axis indicates the distance from the 3′end. Regarding qPCR primers for detection, the primers for Dap and Thrwere each designed in 12 regions from 5′ to 3′. dT: an oligo dT primer,N6: a random hexamer primer, and N6Z: a ZNA-random hexamer primer. Theerror bar indicates a standard deviation (n=3).

FIG. 4 shows that an RT-RamDA method acts with thermally unstabledsDNase and RNase H minus reverse transcriptase. In FIG. 4A, thedetected amount of cDNA in the case of using, as a template, 10 pg oftotal RNA derived from mouse ES cells is shown as a relativequantitative value using qPCR. The value indicates a relative value tothe amount of cDNA (copy number) detected in each gene under RamDA (−)conditions. The error bar indicates a standard deviation (n=4). FIG. 4Bshows the agarose gel electrophoresis patterns of cDNAs synthesized byusing, as templates, 20 ng of MillenniumRNA Markers (0.5, 1, 1.5, 2,2.5, 3, 4, 5, 6, and 9 kb) and by employing various types of reversetranscriptase kits. The alphabets a to j correspond to a to j in thegraph shown in FIG. 4C. A band forming an RNA-DNA hybrid strand isstained brightly than single-stranded cDNA (Non Patent Literature 21).The elongation reaction was carried out at 42° C. for 2.5 minutes. InFIG. 4C, the detected amount of cDNA in the case of using, as atemplate, 10 pg of total RNA derived from mouse ES cells is shown as arelative quantitative value using qPCR. In the figure, the symbol “−”indicates RamDA (−), and the symbol “+” indicates RamDA (+). The valueof RamDA (−) for each enzyme was defined as 1. In RamDA (+) for each ofa to c, h and i, amplification was observed at a magnification of 2times or more. The error bar indicates a standard deviation (n=4).

FIG. 5 shows that RamDA-B and RamDA-C exhibit extremely highamplification rates in specific genes. FIG. 5A shows the amount of cDNAdetected by qPCR using, as a template, 10 pg of total RNA derived frommouse ES cells (upper case) and the relative value obtained when RamDA(−) is set at 1 (lower case). The error bar indicates a standarddeviation (n=4). FIG. 5B shows the effects of thermal cycling. Thefigure shows the amount of cDNA detected by qPCR using, as a template,10 pg of total RNA derived from mouse ES cells (upper case) and therelative value obtained when RamDA (−) under isothermal conditions isset at 1 (lower case). The error bar indicates a standard deviation(n=3). In the figure, “RamDA (−) iso” and “RamDA (+) iso” indicateisothermal conditions (the same conditions as those of RamDA-A), and“RamDA (−) cycle” and “RamDA (+) cycle” indicate thermal cyclingconditions.

FIG. 6 shows a decrease in the nuclease activity of DNase I in aTris-HCl and KCl concentration-dependent manner. According to FRETanalysis using a fluorescent oligonucleotide, the nuclease activity ofDNase I was measured. With regard to the fluorescent oligonucleotide,the fluorescent amount was measured using one color of FAM label probeat 37° C. every 1 minute. The nuclease activity was calculated based onthe fluorescence increase rate in the initial stage of reaction (5-10minutes). The activity was indicated as a relative value, when theactivity to double-stranded DNA in a DNase I buffer (50 mM Tris-HCl, 75mM KCl, and 2 mM MgCl2) was set at 1. The value was calculated based onthe mean value of n=3. The symbol # indicates the concentration of aDNase I buffer. The symbol ## indicates the concentration of Tris-HCland KCl that is the same as that of a common reverse transcriptionbuffer. dsDNA: double-stranded DNA; DNA in RNA: DNA hybrid: DNA in ahybrid strand of RNA and DNA; ssDNA: single-stranded DNA; and ssRNA:single-stranded RNA.

FIG. 7 shows that an RT-RamDA method of using DNase I (RamDA-D) is notlimited to a specific reaction buffer composition. The yield of cDNA inan RT-RamDA method of using DNase I and also using, as a template, 10 pgof total RNA derived from mouse ES cells, was quantified by qPCR. Thesymbol # indicates the same reaction buffer composition as that of aDNase I buffer. The symbol ## indicates the concentration of Tris-HCland KCl that is the same as that of a common reverse transcriptionreaction buffer. The symbol ### indicates the same composition as thatof a common reverse transcription reaction buffer, such as FirstStrandbuffer. FS: First-Strand Buffer. PS: PrimeScript Buffer (for Real Time).Setting RamDA (−) of using PrimeScript Buffer (for Real Time) as acontrol (*), the yield of cDNA is indicated as a relative value, whenthe amount of cDNA detected in each gene in the control is set at 1. Theerror bar indicates a standard deviation (n=4). Amplification takesplace even in the same composition (#) as that of a DNase I bufferhaving high activity of DNase I to single-stranded DNA. Rather, theamplification rate is higher than under conditions in which the activityto single-stranded DNA is low (##).

FIG. 8 shows that a T4 gene 32 protein suppresses fragmentation of cDNAby DNase I. In FIG. 8A, using 5 ng of Thr RNA (2,052 b) as a template,cDNA distribution patterns were compared under conditions of a non-addedstandard reverse transcription sample, single addition of a T4 gene 32protein, single addition of DNase I or dsDNase, and simultaneousaddition of a T4 gene 32 protein with DNase I or dsDNase. The reversetranscription reaction was carried out in a reaction volume of 6 μl thatis two times of ordinary amount, using only an oligo dT primer. Thedistribution pattern of cDNA was examined by 2% agarose gelelectrophoresis. FIG. 8B shows the results obtained by analyzing thecDNA of FIG. 8A that was in an amount of 1/20 of FIG. 8A, usingBioAnalyzer RNA 6000 Pico Kit (Agilent Biotechnology). The numericalvalues in the figure each show a relative value of the cDNAconcentration. T4GP32: T4 gene 32 protein.

FIG. 9 shows that a T4 gene 32 protein contributes to amplification ofcDNA and the stability thereof. The figure shows the amount of cDNA in100 fg of artificially synthesized RNAs, Dap RNA (96,725 copies) and ThrRNA (89,991 copies), which were added to 10 pg of Universal HumanReference RNA (UHRR). The reverse transcription reaction was carried outusing only an oligo dT primer, and the yield of cDNA was quantifiedusing qPCR. DNase I and dsDNase were used in amounts of 0.2 U and 0.4 U,respectively, and the T4 gene 32 protein was used in an amount of 100 ngfor a single reaction. The qPCR was carried out using the reversetranscription product in an amount of 1/50. Regarding qPCR primers fordetection, the primers for Dap and Thr were each designed in 12 regionsfrom 5′ to 3′. FIG. 9A shows the amount of cDNA (copy number) detectedin each region. The horizontal axis indicates the distance from the 3′end. (−): control (a non-added standard reverse transcription sample),and the error bar indicates a standard deviation (n=3). FIG. 9B shows arelative value, when the detected amount in each region of the non-addedcontrol sample (*) is set at 1. FIG. 9C shows a fluctuation in therelative values (amplification rates) of the detection amounts on thesame RNA. T4GP32: T4 gene 32 protein.

FIG. 10 shows the effect of a T4 gene 32 protein on nuclease activity ina reverse transcription reaction buffer, which was examined by FRETanalysis using fluorescent oligonucleotides. As such fluorescentoligonucleotides, two colors, namely, FAM or HEX label probe, were used,and the fluorescence intensity was measured at 37° C. every 1.5 minutes.The nuclease activity was calculated based on the fluorescence increaserate in the initial stage of reaction (1.5-9 minutes) (a mean value ofn=3). As a reserve transcription buffer, PrimeScript buffer was used.FIG. 10A indicates a relative value of nuclease activity, when theactivity of DNase I on double-stranded DNA in a DNase I buffer is setat 1. FIG. 10B shows the ratio of single-stranded DNA-degrading activityto DNA-degrading activity in an RNA-DNA hybrid, or to double-strandedDNA-degrading activity, in individual reaction conditions. dsDNA:double-stranded DNA; DNA in hybrid: DNA in the RNA-DNA hybrid; ssDNA:single-stranded DNA; and ssRNA: single-stranded RNA.

FIG. 11 shows an increase in the yield of cDNA in a reactiontime-dependent manner, in RamDA-D of using a 1-cell lysate of mouse EScells. Among the reaction temperature conditions for RamDA-D, 37° C. wasapplied, the reaction time at 37° C. was prolonged to 30 minutes, 60minutes, and 120 minutes, and the influence of the reaction time on theamplification rate was then examined. Using the reaction time of 30minutes in RamDA-D (−) as a control (*), a relative value is shown, whenthe amount of cDNA detected in each gene by qPCR is set at 1. The errorbar indicates a standard deviation (n=4).

FIG. 12 shows that, in RamDA-D, amplification can be carried out, evenif the type of reverse transcriptase is not limited and template RNA isused in an amount corresponding to 100 cells. Setting the RamDA (−)condition of PrimeScript as a control (*), a relative value is shown,when the amount of cDNA detected in each gene by qPCR is set at 1. Astemplate RNA, total RNA derived from mouse ES cells (10 pg, 200 pg, or 1ng) was used. PS: PrimeScript RT Enzyme; SSII: SuperScript II RT Enzyme;SSIII: SuperScript III RT Enzyme; and SSIV: SuperScript IV RT Enzyme.The error bar indicates a standard deviation (n=4).

EMBODIMENTS FOR CARRYING OUT THE INVENTION

Hereinafter, the present invention will be more specifically described.

The method for amplifying a nucleic acid according to the presentinvention is carried out by incubating a mixture comprising templateRNA, a primer, a degrading enzyme specific to DNA in RNA-DNA hybrid anRNase H minus reverse transcriptase, and a substrate, and this method isalso referred to as Reverse Transcription with Random DisplacementAmplification (RT-RamDA) Method. The method for amplifying a nucleicacid of the present invention is a reverse transcription method withrandom displacement amplification, in which a strand displacementreaction is utilized and RNA is directly used as a template, and thismethod can be carried out in vitro. The method of the present inventionis a technique of amplifying cDNA in a reverse transcription reaction,whereby the cDNA strand in the RNA-cDNA hybrid is cleaved by utilizingthe nuclease activity of a degrading enzyme specific to DNA in RNA-DNAhybrid as endonuclease, such as a double strand-specific DNA degradingenzyme (double-strand specific DNase: dsDNase) or a non-specific DNAdegrading enzyme (DNase I), so that a strand displacement reaction isallowed to randomly take place.

In the present description, the term “amplification” is used to mean toincrease the number of copies (replications) of the sequence of anucleic acid. In addition, examples of the form of amplification includelinear amplification, real-time amplification, quantitativeamplification, semi-quantitative amplification, and competitiveamplification, but are not particularly limited thereto.

The mixture used in the present invention is a mixture comprisingtemplate RNA, a primer, a degrading enzyme specific to DNA in RNA-DNAhybrid, an RNase H minus reverse transcriptase, and a substrate. Thepresent mixture is preferably an aqueous buffer, and more preferably anaqueous buffer comprising salts.

An outline of one aspect of the RT-RamDA method of the present inventionwill be described below (FIG. 1).

-   1. The complementary DNA (cDNA) of template RNA is synthesized by    the RNA-dependent DNA polymerase activity of an RNase H minus    reverse transcriptase.-   2. A cleavage point (nick) is randomly made in the cDNA strand in    the RNA-cDNA hybrid by the nuclease activity of a degrading enzyme    specific to DNA in RNA-DNA hybrid, such as a double strand-specific    DNA degrading enzyme (dsDNase) or a non-specific DNA degrading    enzyme (DNase I).-   3. The nick site becomes a starting point, and the cDNA strand on    the 3′ side is peeled by the strand displacement activity of the    RNase H minus reverse transcriptase. cDNA is newly synthesized in    the peeled portion by reverse transcriptase. The peeled cDNA is    protected from the nuclease activity by a T4 gene 32 protein    (T4GP32) as a single-stranded DNA-binding protein. When this    phenomenon takes place continuously, the yield of cDNA can be    increased to 10-fold or more.

Since the RNA-DNA hybrid strand is targeted by the degrading enzymespecific to DNA in RNA-DNA hybrid in the method for amplifying a nucleicacid of the present invention, or since the template is RNA in thestrand displacement amplification reaction, the available reversetranscriptase is limited to those having no RNase H activity. The RNaseH activity is ribonuclease activity of randomly cleaving only the RNAstrand in an RNA-DNA hybrid. RNase H is non-specific endonuclease, whichcatalyzes RNA cleavage by hydrolysis.

Moreover, the RNase H minus reverse transcriptase used in the presentinvention has a strand displacement activity. The cDNA strand on the 3′side is peeled from RNA by the strand displacement activity of the RNaseH minus reverse transcriptase, and a new cDNA strand is synthesized inthe peeled portion.

The reverse transcription is a process in which reverse transcriptase(RNA-dependent DNA polymerase) catalyzes the formation of complementaryDNA (cDNA) from template RNA. As such reverse transcriptase, a largenumber of enzymes have been identified, and examples of the reversetranscriptase include HIV reverse transcriptase, AMV reversetranscriptase, M-MLV reverse transcriptase, C therm. polymerase, and Tthpolymerase. In the present invention, RNase H minus reversetranscriptase is used.

In the present invention, a degrading enzyme specific to DNA in RNA-DNAhybrid is used. The term “degrading enzyme specific to DNA in RNA-DNAhybrid” is used in the present description to mean an enzyme having anactivity of cleaving a DNA strand in a hybrid strand of RNA and DNA(RNA-DNA hybrid). As such a degrading enzyme specific to DNA in RNA-DNAhybrid, a double strand-specific DNA degrading enzyme or a non-specificDNA degrading enzyme can be used. As a double strand-specific DNAdegrading enzyme, a crab-derived double strand-specific DNA degradingenzyme (double strand-specific nuclease; DSN) has been known. Thisenzyme randomly cleaves not only double stranded DNA, but also only theDNA strand in an RNA-DNA hybrid. In the present invention, there hasbeen proposed a method of utilizing the aforementioned activity tocleave the cDNA in an RNA-cDNA hybrid formed during reversetranscription, so as to cause strand displacement amplification whileusing RNA as a template. However, since the temperature for activationof crab-derived DSN is an extremely high temperature that is 60° C. orhigher, it is unsuitable for a general reverse transcription reaction(Non Patent Literature 11). Hence, by using shrimp-derived heat-labiledsDNase that is active even at a low temperature, both a reversetranscription reaction and cleavage with DNase could be achieved (NonPatent Literature 12). On the other hand, when a non-specific DNAdegrading enzyme such as DNase I is used, single-stranded DNA, namely,amplified DNA that is preceded by a strand displacement reaction or areverse transcription primer is likely to be decomposed. However, evenif the enzyme has an activity of degrading single-stranded DNA, it is acase where it would not cause a problem, depending on reactionconditions such as the composition of a buffer used. Whether or not theenzymes used or reaction conditions are suitable can be confirmed byperforming a nucleic acid amplification reaction according to thedisclosure of the present description. Moreover, by addition of asingle-stranded DNA-binding protein, the degradation activity ofsingle-stranded DNA is suppressed, and the synthesis and amplificationof cDNA can be promoted by a strand displacement reaction using reversetranscriptase. As such a single-stranded DNA-binding protein, any givenprotein, which binds to single-stranded DNA to increase the efficiencyof nucleic acid amplification according to the method of the presentinvention, can be used, and for example, any given origin-derived T4gene 32 protein, RecA, SSB (Single-Stranded DNA Binding Protein), or avariant thereof can be used. Thereby, the amplification rate can beincreased, rather than by a method of using dsDNase. The use of dsDNaseor DNase I is advantageous in that DNA contaminated with the reactionsolution, for example, genomic DNA in a cell lysate sample can also beremoved. Also in the past, there have been reported methods of removingcontaminated DNA using DNase I or dsDNase in RT-qPCR. However, in amajority of such methods, inactivation of nuclease has been essential,and thus, such methods could not be carried out by only one stepconsisting of a reverse transcription reaction, like the RT-RamDA method(Non Patent Literature 13).

There have been many reports regarding DNA amplification methods using astrand displacement reaction, as with the RT-RamDA method (for example,Non Patent Literatures 2 to 6). However, a majority of theseamplification methods including SDA as a typical example need to userestriction enzymes to insert a nick into template DNA, or need to use aplurality of sequence-specific primers in combination. Accordingly, itis essential to add a restriction enzyme recognition sequence ordeoxyinosine to template DNA, or to design and/or synthesize asequence-specific primer. Moreover, target genes are also limited.Furthermore, since almost all of existing methods use DNA as a template,it is necessary to perform a reverse transcription reaction ofconverting RNA to DNA in order to amplify an RNA sequence. Further,pseudo-positive results generated as a result of the carry-over of theprevious PCR amplification products or reverse transcription products onsamples, and non-specific products derived from DNA contaminated withreagents, DNA mixed from working environment, etc., have beenproblematic (Non Patent Literatures 9 and 10). In particular, in MDA,since a primer is randomly annealed to DNA, it is highly likely that thecontaminated DNA-derived non-specific products would be increased. Onthe other hand, the RT-RamDA method of the present invention uses anon-sequence-dependent strand displacement reaction, and thus, itbecomes unnecessary to design and/or synthesize a sequence-specificprimer or a special primer, and also, it can target whole genes.Furthermore, by directly using RNA as a template, the process fromreverse transcription to amplification can be completed by only onestep, and since DNA hardly becomes a template, amplification ofnon-specific products derived from the contaminated DNA can also bereduced.

The double strand-specific DNA degrading enzyme used in the presentinvention can be preferably an enzyme, which has an activity of cleavingthe DNA strand of an RNA-DNA hybrid, and substantially does not have anactivity of cleaving the RNA strand of an RNA-DNA hybrid,single-stranded DNA and single-stranded RNA. The double strand-specificDNA degrading enzyme is preferably an enzyme having a DNA-degradingactivity even at a temperature of lower than 60° C.

As such double strand-specific DNA degrading enzyme, enzymes derivedfrom prokaryotes or eukaryotes can be used. Preferably, aCrustacea-derived double strand-specific DNA degrading enzyme or avariant thereof can be used.

As such double strand-specific DNA degrading enzyme (which is alsoreferred to as “double strand-specific nuclease (DSN)”), the followingenzymes have been known so far (JP Patent Publication (Kokai)2014-103867 A).

-   (1) Solenocera melantho (coastal mud shrimp) DNase-   (2) Penaeus japonicus (prawn) DNase-   (3) Paralithodes camtschaticus (king crab) DSN-   (4) Pandalus borealis (Northern shrimp) dsDNase-   (5) Chionoecetes opilio (snow crab) DSN-   (6) Other DSN homologs

Among the above-described enzymes, king crab DSN and snow crab DSN haveheat resistance at an optimum active temperature of about 60° C.,whereas Northern shrimp dsDNase is a heat-labile enzyme having anoptimum temperature of 37° C.

In the present invention, a shrimp-derived double strand-specific DNAdegrading enzyme or a variant thereof is further preferable.

In the present description, the term “variant” is used to mean an enzymeobtained by modifying the amino acid sequence of a naturalproduct-derived double strand-specific DNA degrading enzyme.Specifically, such a variant means an enzyme consisting of an amino acidsequence having sequence identity of 80% or more (preferably 90% ormore, and more preferably 95% or more) to the amino acid sequence of anatural product-derived double strand-specific DNA degrading enzyme, andhaving a double strand-specific DNA-degrading activity, and also, anenzyme consisting of an amino acid sequence comprising a deletion,substitution and/or addition of one or several amino acids (for example,1 to 10, preferably 1 to 5, and more preferably 1 to 3 amino acids) withrespect to the amino acid sequence of a natural product-derived doublestrand-specific DNA degrading enzyme, and having a doublestrand-specific DNA-degrading activity.

As a double strand-specific DNA degrading enzyme, a commerciallyavailable product can be used. Examples of such a commercially availableproduct include dsDNase (ArcticZymes), Hl-dsDNase (ArcticZymes), dsDNase(Thermo scientific), Shrimp DNase, Recombinant (Affymetrix), AtlantisdsDNase (Zymo Research), and Thermolabile Nuclease (Roche).

An example of the non-specific DNA degrading enzyme used in the presentinvention is an enzyme, which has an activity of cleaving the DNA strandof an RNA-DNA hybrid and substantially does not have an activity ofcleaving the RNA strand of an RNA-DNA hybrid and single-stranded RNA,wherein the activity of cleaving the single-stranded DNA is preferablylower than the activity of cleaving the DNA strand of the RNA-DNAhybrid. The non-specific DNA degrading enzyme is preferably an enzymehaving a DNA-degrading activity even at a temperature of lower than 60°C.

As such a non-specific DNA degrading enzyme, an enzyme derived fromprokaryotes or eukaryotes can be used. Preferably, a mammal-derivednon-specific DNA degrading enzyme or a variant thereof can be used.

In the present invention, a bovine-derived non-specific DNA degradingenzyme or a variant thereof is more preferable.

In the present description, the term “variant” is used to mean an enzymeobtained by modifying the amino acid sequence of a naturalproduct-derived non-specific DNA degrading enzyme. Specifically, thevariant means an enzyme consisting of an amino acid sequence havingsequence identity of 80% or more (preferably 90% or more, and morepreferably 95% or more) to the amino acid sequence of a naturalproduct-derived non-specific DNA degrading enzyme, and having anon-specific DNA-degrading activity, and also, an enzyme consisting ofan amino acid sequence comprising a deletion, substitution and/oraddition of one or several amino acids (for example, 1 to 10, preferably1 to 5, and more preferably 1 to 3 amino acids) with respect to theamino acid sequence of a natural product-derived non-specific DNAdegrading enzyme, and having a non-specific DNA-degrading activity.

The primer used in the present invention is composed of adeoxynucleotide and/or a ribonucleotide, and the present primer has achain length capable of undergoing base pairing with a target nucleicacid under given conditions. The chain length of such a primer is notparticularly limited, and it is preferably 5 to 50 nucleotides, and morepreferably 5 to 30 nucleotides. As such primers, one or more of types ofrandom primers, oligo dT primers or sequence-specific primers can bepreferably used. In the case of using a random primer, the length of theprimer is preferably approximately 5 to 10 nucleotides, and morepreferably approximately 6 to 8 nucleotides. In the case of using anoligo dT primer, the length of the primer is preferably 10 to 50nucleotides, and more preferably 15 to 30 nucleotides. In the case ofusing a sequence-specific primer, the length of the primer is preferably5 to 30 nucleotides, and more preferably 7 to 20 nucleotides.

The primer can be synthesized by any given method that can be used inthe synthesis of an oligonucleotide, such as a phosphite triestermethod, an H-phosphonate method, or a thiophosphonate method. The primeraccording to the present invention can be synthesized, for example, by aphosphoramidite method, using DNA Synthesizer Type 394 manufactured byABI (Applied Biosystem Inc.).

Preferably, a primer, the Tm value of which has been increased bymodification with a cation unit, can be used. For example, a Zip NucleicAcid (ZNA) primer can be used. ZNA has an action to increase the Tmvalue of an oligonucleotide, using a cation unit (Non Patent Literatures14 to 16). By using a primer, the Tm value of which has been increasedby modification with a cation unit, such as a ZNA primer, amplificationefficiency can be enhanced. In particular, by using a random hexamerprimer modified with ZNA, the primer can be annealed to template RNAeven at a reaction temperature during reverse transcription, andthereby, more efficient strand displacement amplification can be carriedout.

The term “substrate” is used in the present description to mean asubstrate of RNase H minus reverse transcriptase, and as such asubstrate, a mixture of four types of deoxyribonucleotides (dATP, dCTP,dGTP, and dTTP) can be used. However, the substrate is not limited tothe mixture of dATP, dCTP, dGTP, and dTTP, and dideoxynucleotidetriphosphate may be added to the mixture, or a modifieddeoxyribonucleotide may also be used.

In the present invention, the mixture to be used in the reaction maycomprise a single-stranded DNA-binding protein such as a T4 gene 32protein. The T4 gene 32 protein can be used as an auxiliary factor forincreasing the uniformity of amplification. The T4 gene 32 protein as asingle-stranded DNA-binding protein has been known to act, not only onsingle-stranded DNA, but also on RNA (Non Patent Literatures 17 to 20).By using the T4 gene 32 protein, the higher structure of template RNAcan be loosened, and a more uniform strand displacement reaction can becarried out on the full-length template. Moreover, in the case of areaction using a non-specific DNA degrading enzyme, decomposition ofamplified cDNA can be prevented.

The nucleic acid amplification reaction according to the presentinvention may be carried out under isothermal conditions, or underthermal cycling conditions.

When the nucleic acid amplification reaction is carried out underisothermal conditions, the reaction can be carried out, for example, ata predetermined temperature between 25° C. and 50° C., preferably at apredetermined temperature between 30° C. and 45° C., and more preferablyat a predetermined temperature between 35° C. and 40° C., and thus, thereaction can be carried out, for example, at 37° C. for a predeterminedperiod of time (for example, within 5 minutes to 3 hours, and preferably10 minutes to 150 minutes). When the reaction is carried out, forexample, at 37° C., after the reaction solution has been incubated, forexample, at 25° C. for a predetermined period of time (for example, 5minutes to 15 minutes), and then, has been incubated at 30° C. for apredetermined period of time (for example, 5 minutes to 15 minutes), thereaction is carried out at 37° C. Alternatively, after the reactionsolution has been incubated at 37° C., it may be incubated at 50° C. fora predetermined period of time (for example, 5 minutes to 15 minutes),and then at 85° C. for a predetermined period of time (for example, 5minutes to 15 minutes).

When the nucleic acid amplification reaction is carried out underthermal cycling conditions, for example, a predetermined temperature T1of 20° C. or higher and lower than 30° C. (for example, 25° C.) iscombined with a predetermined temperature T2 of 30° C. or higher and 45°C. or lower (for example, 37° C.). Then, T1 for a predetermined periodof time (for example, 1 minute to 3 minutes, and as an example, 2minutes) and T2 for a predetermined period of time (for example, 1minute to 3 minutes, and as an example, 2 minutes) are set at one cycle,and this cycle is repeated preferably for 10 cycles to 40 cycles, andmore preferably for 15 cycles to 35 cycles, so that the reaction can becarried out. In addition, prior to the above-described thermal cycling,the reaction solution may be incubated, for example, at 25° C. for apredetermined period of time (for example, 5 minutes to 15 minutes),then at 30° C. for a predetermined period of time (for example, 5minutes to 15 minutes), and then at 37° C. for a predetermined period oftime (for example, 1 minute to 5 minutes). Moreover, after completion ofthe above-described thermal cycling, the reaction solution may beincubated at 50° C. for a predetermined period of time (for example, 5minutes to 15 minutes), and then at 85° C. for a predetermined period oftime (for example, 5 minutes to 15 minutes).

The method for amplifying a nucleic acid according to the presentinvention can be used as a part of an RT-qPCR method of using a traceamount of RNA (for example, trace RNA corresponding to a volume rangingfrom a single cell to several hundreds of cells).

The method for amplifying a nucleic acid according to the presentinvention can be utilized in an RNA sequence method of using a traceamount of RNA (for example, trace RNA corresponding to a volume rangingfrom a single cell to several hundreds of cells).

By utilizing the method for amplifying a nucleic acid according to thepresent invention, it is possible to detect a single cell in a largeamount of cells. Specifically, by specifically amplifying genesexpressing only in target cells contained in a large amount of cells, itbecomes possible to detect only a few target cell genes contained in alarge amount of RNA, so that the presence or absence of target cells canbe judged.

The present invention further relates to a kit for carrying out theabove-described method for amplifying a nucleic acid according to thepresent invention. The kit of the present invention comprises, at least,a DNA strand-specific RNA-DNA hybrid strand degrading enzyme such as adouble strand-specific DNA degrading enzyme or a non-specific DNAdegrading enzyme, and an RNase H minus reverse transcriptase.Preferably, the kit of the present invention further comprises asingle-stranded DNA-binding protein (for example, a T4 gene 32 protein).The kit of the present invention may further comprise, as desired, otherreagents necessary for carrying out the nucleic acid amplificationreaction, a buffer, and the like. Examples of such other reagentsinclude a primer, and deoxyribonucleotide triphosphate.

The present invention will be described in more detail in the followingexamples. However, these examples are not intended to limit the scope ofthe present invention.

EXAMPLES

The RT-RamDA method is a method for amplifying a nucleic acid, whichcomprises a step of incubating a mixture comprising template RNA, aprimer, a degrading enzyme specific to DNA in RNA-DNA hybrid such as adouble strand-specific DNA degrading enzyme or a non-specific DNAdegrading enzyme, an RNase H minus reverse transcriptase, and asubstrate. In the RT-RamDA method, the complementary strand DNA (cDNA)of template RNA is synthesized by the RNA-dependent DNA polymeraseactivity of the RNase H minus reverse transcriptase, the cDNA strand inthe RNA-cDNA hybrid is randomly cleaved with the degrading enzymespecific to DNA in RNA-DNA hybrid. The above described cleavage sitebecomes a starting point, the cDNA strand on the 3′ side is peeled fromRNA by the strand displacement activity of the RNase H minus reversetranscriptase, and a novel cDNA strand is synthesized in the portionpeeled by the RNase H minus reverse transcriptase.

RamDA-A is an aspect of carrying out the RT-RamDA method, using a doublestrand-specific DNA degrading enzyme and a T4 gene 32 protein.

RamDA-B is an aspect of carrying out the RT-RamDA method, using a doublestrand-specific DNA degrading enzyme, without using a T4 gene 32protein.

RamDA-C is an RT-RamDA thermal cycling method, which is an aspect ofperforming a reaction by modifying reaction temperature conditions,while maintaining the same composition of the reaction solution as thatin RamDA-B, and by repeating with short intervals an annealing step andan elongation reaction step.

RamDA-D is an aspect of carrying out the RT-RamDA method, using anon-specific DNA degrading enzyme and a T4 gene 32 protein.

(Materials and Methods) Cell Culture

For extraction of total RNA, 5G6GR mouse ES cells were used. This cellline was produced by randomly incorporating a linearizedGata6-GR-IRES-Puro vector into EB5 ES cells (Non Patent Literature 20).The cells were cultured on a feeder-free gelatin-coated dish, in a 10%fetal bovine serum-containing Glasgow minimal essential medium (GMEM;Sigma-Aldrich, St. Louis, Mo., USA) comprising 1000 U/ml leukemiainhibitory factor (ESGRO; Invitrogen Corp., Carlsbad, Calif., USA), 100μmol/1 2-mercaptoethanol (Nacalai Tesque Inc., Kyoto, Japan),1×non-essential amino acids (Life Technologies Corp., Carlsbad, Calif.,USA), 1 mmol/1 sodium pyruvate (Life Technologies), 2 mmol/1 L-glutamine(Nacalai Tesque), 0.5×penicillin/streptomycin (Life Technologies), and10 μg/ml blasticidin (Life Technologies).

RNA Extraction

Total RNA was purified using RNeasy Mini Kit (Qiagen Inc., Valencia,Calif., USA). Quantification of RNA and the quality check thereof werecarried out using Quantus Fluorometer (Promega Corp., Madison, Wis.,USA) and RNA 6000 Nano Kit (Agilent Biotechnology, Santa Clara, Calif.,USA). Artificially synthesized RNA was synthesized from pGIBS-DAP,pGIBS-THR plasmid (American Type Culture Collection (ATCC), Manassas,Va., USA), using MEGAscript T3 kit (Life Technologies).

Preparation of 1-cell lysate of Mouse ES Cells

The cultured cells were reacted at 37° C. for 3 minutes, using TrypLEExpress (Life Technologies), so as to dissociate the cells into 1 cell.After completion of the dissociation, the reagent was immediatelyreplaced with PBS (−) to terminate the reaction. In order to preciselyfractionate only the cell using a cell sorter, the living cell nucleusof the dissociated cell was labeled by staining it using Vybrant DyeCycle (Life Technologies). The method included in the reagent wasapplied as staining conditions. Using SH 800 Cell Sorter (Sony Corp.,Tokyo, Japan), cells, which showed positive to Vybrant Dye Cycle(detection filter: BP450/50) and showed negative to dead cellfluorescence marker dye, PI (detection filter: BP585/40), were definedas a living cell fraction. From this fraction, 50 cells werefractionated into 1 μl of Lysis buffer (1 U of RNasein plus (Promega),0.3% NP40 (Thermo Fisher Scientific Inc., Waltham, Mass. USA), and RNasefree water (TaKaRa Bio Inc., Otsu, Japan)). After completion of thefractionation, the cells were immediately subjected to spinning down andstirring by shaking, and the resulting cells were then conserved at −80°C. When the cells were used in a reverse transcription reaction, thecells were thawn, and 49 μl of Lysis buffer was then added to theresulting cells, so that 1 μl of 50-fold diluted cell lysate was used asa 1-cell lysate.

FRET Assay

FRET analysis was carried out by modifying the method of Inge W. Nilsenet al (Non Patent Literature 12). 0.6 μl of oligonucleotide probe mix (6pmol of oligo DNA or RNA (Sigma-Aldrich), 1×First-Strand buffer (LifeTechnologies), and RNase free water (TaKaRa)) was prepared, and was thendenatured at 70° C. for 5 minutes. Thereafter, the temperature wasslowly returned to room temperature. Combinations of oligonucleotidesused in the analysis are as follows (SEQ ID NOS: 1 to 10).

Dual probe for double-stranded DNA: FAM-CGCCATCGGAGGTTC-BHQ1HEX-GAACCTCCGATGGCG-BHQ1 Dual probe for RNA-DNA hybrid:FAM-CGCCATCGGAGGTTC-BHQ1 HEX-rGrArArCrCrUrCrCrGrArUrGrGrCrG-BHQ1Single probe for single-stranded DNA: HEX-GAACCTCCGATGGCG-BHQ1Single probe for single-stranded RNA:HEX-rGrArArCrCrUrCrCrGrArUrGrGrCrG-BHQ1 Single FAM probe for correction:FAM-CGCCATCGGAGGTTC-BHQ1 GAACCTCCGATGGCGSingle HEX probe for correction: CGCCATCGGAGGTTCHEX-GAACCTCCGATGGCG-BHQ1

Subsequently, 5.4 μl of enzyme solution (1×First-Strand buffer, 0.12 Uof each nuclease, and RNase free water) was prepared. As nucleases,DNase I Amplification Grade (Life Technologies), dsDNase (ArcticZymesAS, Tromso, Norway), and HL-dsDNase (ArcticZymes) were used. As acontrol, RNase free water was added instead of the enzyme. 0.6 μl of theoligonucleotide probe mix, which had been returned to room temperature,was mixed with 5.4 μl of the enzyme solution on PloxiPlate-384F Plus(PerkinElmer Inc., Waltham, Mass., USA), and the obtained mixture wasthen measured using EnVision (PerkinElmer). For the measurement, thefluorescence intensities of FAM and HEX were measured at 37° C. at arate of one measurement for 70 seconds, and this measurement was carriedout 50 times. The leakage of the fluorescence of FAM and HEX wascorrected using the fluorescence amount of each single probe forcorrection.

In a verification experiment regarding the action of a T4 gene 32protein on nuclease activity (FIG. 10), 0.6 μl of oligonucleotide probemix (6 pmol of oligo DNA or RNA (Sigma-Aldrich), 1×PrimeScript Buffer(for Real Time) (TaKaRa), and RNase free water) and 5.4 μl of enzymesolution (1×PrimeScript Buffer (for Real Time) or 1×DNase I reactionbuffer (Life Technologies), 0.12 U of each nuclease, and RNase freewater) were prepared. As nucleases, DNase I Amplification Grade anddsDNase were used. As a T4 gene 32 protein-added sample, 180 ng of T4gene 32 protein (Roche Applied Science, Indianapolis, Ind., USA) wasadded to the enzyme solution. As a control, RNase free water was addedinstead of the enzyme. The measurement was carried out at a rate of onemeasurement for 90 seconds, 50 times. The enzyme activity was calculatedusing the fluorescence amplification rate in the initial stage ofreaction (1.5 minutes to 9 minutes).

In a measurement experiment regarding KCl and Tris-HClconcentration-dependent nuclease activity of DNase I (FIG. 6), 0.6 μl ofoligonucleotide probe mix (6 pmol of oligo DNA or RNA (Sigma-Aldrich),1×DNase I reaction buffer (Life Technologies), and RNase free water) and5.4 μl of enzyme solution (in-house reaction buffer, 0.12 U of DNase IAmplification Grade, and RNase free water) were prepared. The in-housereaction buffer was adjusted to have a final concentration shown in FIG.6, depending on conditions. The measurement was carried out at a rate ofone measurement for 60 seconds, 50 times. The enzyme activity wascalculated using the fluorescence amplification rate in the initialstage of reaction (5 minutes to 10 minutes). Combinations ofoligonucleotides used in the analysis are as follows (SEQ ID NOS: 11 to16).

Single probe for double-stranded DNA: FAM-CGCCATCGGAGGTTC-BHQ1GAACCTCCGATGGCG-BHQ1 Single probe for RNA-DNA hybrid:FAM-CGCCATCGGAGGTTC-BHQ1 rGrArArCrCrUrCrCrGrArUrGrGrCrG-BHQ1Single probe for single-stranded DNA: FAM-CGCCATCGGAGGTTC-BHQ1Single probe for single-stranded RNA:FAM-rCrGrCrCrArTrCrGrGrArGrGrTrTrC-BHQ1

-   Reverse Transcription Reaction    -   RT-RamDA Method (RamDA-A):

Template RNA was diluted with 1 μl of Lysis buffer (1 U of RNasein plus(Promega), 10% Roche lysis buffer (Roche), 0.3% NP40 (Thermo Fisher),and RNase free water), and was then subjected to a denaturationtreatment at 65° C. for 2 minutes. The resulting RNA was conserved onice before use. ReverTra Ace qPCR RT KIT (TOYOBO Co. Ltd., Osaka, Japan)was modified, and was used as a reverse transcriptasethe reactionsolution. To 1 μl of the denatured diluted template RNA, 2 μl of RT mix(1.5×ReverTra Ace RT buffer (TOYOBO), 0.6 pmol of oligo (dT) 18 primer(Thermo Fisher), 7.8 pmol of random hexamer primer (TaKaRa),1.5×ReverTra Ace enzyme mix (TOYOBO), and RNase free water), or 2 μl ofRamDA-A mix (1.5×ReverTra Ace RT buffer, 0.6 pmol of oligo (dT) 18primer, 7.8 pmol of ZNA-random hexamer primer ([Z][Z]NNNNNN;Sigma-Aldrich), 0.4 U of dsDNase (ArcticZymes), 100 ng of T4 gene 32protein, 1.5×ReverTra Ace enzyme mix, and RNase free water) was added.The thus obtained mixtures were each stirred at 2,000 rpm for 30 secondsusing MixMate (Eppendorf, Westbury, N.Y., USA), and were then reactedusing a thermal cycler C1000 (Bio-Rad Laboratories, Inc., Hercules,Calif., USA)), at 25° C. for 10 minutes, at 30° C. for 10 minutes, at37° C. for 30 minutes, at 50° C. for 5 minutes, and at 85° C. for 5minutes.

-   RT-RamDA Method without Using T4 Gene 32 Protein (RamDA-B):

Template RNA was diluted with 1 μl of lysis buffer (1 U of RNasein plus,0.3% NP40, and RNase free water), and was then subjected to adenaturation treatment at 70° C. for 90 seconds. The resulting RNA wascooled on ice, and thereafter, 2 μl of RT mix (1.5×ReverTra Ace RTbuffer, 0.6 pmol of oligo (dT) 18 primer, 7.8 pmol of random hexamerprimer, 1.5×ReverTra Ace enzyme mix, and RNase free water), or 2 μl ofRamDA-B mix (1.5×ReverTra Ace RT buffer, 0.6 pmol of oligo (dT) 18primer, 7.8 pmol of ZNA-random hexamer primer([Z][Z]NNNNNN;Sigma-Aldrich), and 0.1 U of dsDNase) was added. Thereafter, the thusobtained mixtures were each subjected to the same operations as thoseperformed in RamDA-A.

-   RT-RamDA Thermal Cycling Method (RamDA-C):

A reverse transcription reaction was carried out with the same reactionsolution composition as that of RamDA-B, with the exception that thetemperature conditions were changed. The reaction conditions are asfollows. After a reaction had been carried out at 25° C. for 10 minutes,at 30° C. for 10 minutes, and at 37° C. for 2 minutes, a reaction at 25°C. for 2 minutes, and then at 37° C. for 2 minutes was carried out for29 cycles. Thereafter, the reaction product was treated at 50° C. for 5minutes, and then at 85° C. for 5 minutes.

-   RT-RamDA Non-specific DNA Degrading Enzyme (DNase I) Method    (RamDA-D):

Template RNA was diluted with 1 μl of Lysis buffer (1 U of RNasein plus,10% Roche lysis buffer, 0.3% NP40, and RNase free water), and was thensubjected to a denaturation treatment at 70° C. for 90 seconds. Theresulting RNA was conserved on ice before use. PrimeScript RT reagentKit(Perfect Real Time) (TaKaRa) was modified, and was used as a reversetranscriptasethe reaction solution. To 1 μl of the denatured templateRNA, 2 μl of RT mix (1.5×PrimeScript Buffer (for Real Time), 0.6 pmol ofoligo (dT) 18 primer, 8 pmol of random hexamer primer, 1.5×PrimeScriptRT Enzyme Mix I, and RNase free water), or 2 μl of RamDA-D mix(1.5×PrimeScript Buffer (for Real Time), 0.6 pmol of oligo (dT) 18primer, 8 pmol of random hexamer primer, 0.2 U of DNase I, AmplificationGrade, 100 ng of T4 gene 32 protein , 1.5×PrimeScript RT Enzyme Mix I,and RNase free water) was added. The thus obtained mixtures were eachreacted at 25° C. for 10 minutes, at 30° C. for 10 minutes, at 37° C.for 60 minutes, at 50° C. for 5 minutes, and at 85° C. for 5 minutes.

-   Experiment for Studying Composition of Reverse Transcription    Reaction Solution, using RamDA-D:

When a in-house reverse transcription reaction buffer, in which theconcentrations of Tris-HCl, KCl, NaCl and MgCl₂ were changed, orFirst-Strand Buffer was used, these were replaced with PrimeScriptBuffer (for Real Time) in RamDA-D mix, and dNTP Mix (Life Technologies)was further added to the solution to a final concentration of 0.5 mM,followed by performing the reaction.

-   The Composition of Each Buffer is Shown in Table 1 Below.

TABLE 1 Compositions of in-house reaction buffers and commerciallyavailable enzyme-added reaction buffers 1 X Concentration (mM) Tris-HClTris-HCl Buffer pH 8.4 pH 8.3 KCl NaCl MgCl₂ FIG. 7-a 20.0 50.0 0.0 2.0FIG. 7-b 20.0 62.5 0.0 2.0 FIG. 7-c 20.0 75.0 0.0 2.0 FIG. 7-d 35.0 50.00.0 2.0 FIG. 7-e 35.0 62.5 0.0 2.0 FIG. 7-f 35.0 75.0 0.0 2.0 FIG. 7-g50.0 50.0 0.0 2.0 FIG. 7-h 50.0 62.5 0.0 2.0 FIG. 7-i 50.0 75.0 0.0 2.0FIG. 7-j 50.0 75.0 0.0 3.0 FIG. 7-k 50.0 75.0 10.0 3.0 FIG. 7-l 50.075.0 20.0 3.0 DNase I reaction buffer *20.0 *50.0 *2.0 (LifeTechnologies) First-Strand Buffer *50.0 *75.0 *3.0 (Life Technologies)PrimeScript Buffer — — — — (for Real Time) (TaKaRa) *Catalog value; —Unreleased

-   Artificially Synthesized RNA (Dap and Thr) System of Using RamDA-D:

Only 0.6 pmol of oligo (dT) 18 primer was used as a reversetranscription primer in RamDA-D mix, and the composition of the RamDA-Dmix was changed depending on conditions. In addition, the temperatureconditions for the reverse transcription reaction were determined to bethe same conditions as those for RamDA-A.

-   Studies Regarding Reverse Transcriptase in RamDA-D:

With regard to SuperScript II, SuperScript III, and SuperScript IVconditions, 30 U of SuperScript II (Life Technologies), 30 U ofSuperScript III (Life Technologies), and 30 U of SuperScript IV (LifeTechnologies) were used as reverse transcriptase in RamDA-D mix, insteadof PrimeScript RT Enzyme Mix I, and further, 6 U of RNasein plus werealso added. As a reverse transcription reaction buffer, PrimeScriptBuffer (for Real Time) was used for all conditions.

-   Comparison of dsDNase:

Nuclease was replaced with 0.2 U of 43 KDa dsDNase (ArcticZymes), 0.2 Uof 47 KDa dsDNase (Affymetrix Inc., Santa Clara, Calif., USA), and 0.2 Uof duplex specific nuclease (DSN; Evrogen JSC, Moscow, Russia),respectively, under the RamDA-A reaction solution conditions, and acomparison was then made. The reaction temperature conditions weredetermined in accordance with RamDA-A, and the amplification rate ofcDNA was then quantified by qPCR.

-   Comparison of Reverse Transcriptase:

A comparison was made using 10 types of qPCR RT kits, Maxima H MinusFirst Strand cDNA Synthesis Kit (Thermo Fisher), ReverTra Ace qPCR RTKit (TOYOBO), PrimeScript RT reagent Kit (TaKaRa), AffinityScript QPCRcDNA Synthesis Kit (Agilent Biotechnology), QuantiTect Rev.Transcription Kit (Qiagen), GoScript Reverse Transcription System(Promega), iScript Select cDNA Synthesis Kit (Bio-Rad), ProtoScript IIFirst Strand cDNA Synthesis Kit (New England Biolabs, MA, UK),SuperScript III (Life Technologies), and Transcriptor First Strand cDNASynthesis Kit (Roche).

For an elongation activity test, a reverse transcription reaction wascarried out using Millennium RNA Markers (Life Technologies) comprisingpoly-A-added RNAs with various lengths (0.5-9 k bases). A reversetranscription mixture was prepared using reagents supplied to each kit,applying the conditions for formulation of the reagents recommended bythe kit, in 10 μl of a reaction system. However, as a reversetranscription primer, an oligo (dT) 18 primer was used in all of thekits. 1.5 μl of RNA-primer mix (20 ng of Millennium RNA Markers, 25 pmolof oligo (dT) 18 primer, and lysis buffer (RamDA-A conditions)) wasdenatured at 65° C. for 2 minutes, it was then mixed with 8.5 μl ofreverse transcription mix, and the obtained mixture was then reacted at42° C. for 2.5 minutes, and then at 85° C. for 5 minutes. Aftercompletion of the reaction, the reaction mixture was analyzed by agarosegel electrophoresis.

For an adaptation test of the RamDA method, using 10 pg of total RNAderived from mouse ES cells, a reverse transcription reaction wascarried out. For the preparation of a reverse transcription mixture,reagents supplied to each kit were used, and for formulation of thereagents, 3 μl of a reaction system, which was down-scaled underconditions recommended by the kit, was used. However, regarding reversetranscription primers, 0.6 pmol of oligo (dT) 18 primer and 7.8 pmol ofrandom hexamer primer were used under RamDA (−) conditions, whereas 0.6pmol of oligo (dT) 18 primer and 7.8 pmol of ZNA-random hexamer primerwere used under RamDA (+) conditions. Moreover, in the case of RamDA(+), 0.2 U of dsDNase (ArcticZymes) and 100 ng of T4 gene 32 proteinwere further added. The reaction was carried out under reactiontemperature conditions of 25° C., 10 minutes, 30° C., 10 minutes, 42°C., 30 minutes, 50° C., 5 minutes, and 85° C., 5 minutes, and theamplification rate of cDNA was then quantified by qPCR.

Preparation of reagents and the reactions were all carried out on 0.2 mlof Hi-Tube Flat Cap Recovery (TaKaRa), EU Semi-domed 8-cap strip(BIOplastics BV, Landgraaf, Netherlands), or RP, LF, SEMI Sk, cutable,96 well plate (BIOplastics BV).

-   Quantitative Polymerase Chain Reaction (qPCR)

In the case of an artificially synthesized RNA (Dap and Thr) system, areverse transcription reaction product was diluted with nuclease freewater (Qiagen), and the diluted product in an amount of 1/50 was used asa qPCR reaction solution. In the case of a system of 10 pg of Total RNAderived from mouse ES cells, a reverse transcription reaction productwas diluted with nuclease free water, and the diluted product in anamount of 1/6, 1/8, or 1/10, was used as a qPCR reaction solution. TheqPCR was carried out using LightCyclar 480 (Roche) or ABI 7900HT (LifeTechnologies) under the following conditions. 10 μl of qPCR reactionsolution (1×Quantitect SybrGreen master mix (Qiagen), 5 pmol of forwardprimer, 5 pmol of reverse primer, 3 μl of diluted cDNA, and nucleasefree water) was reacted at 95° C. for 15 minutes to activate the enzyme,and thereafter, denaturation at 95° C. for 15 seconds and an elongationreaction at 60° C. for 1 minute were carried out for 40 cycles. Themelting curve was analyzed at 95° C. for 15 seconds, at 60° C. for 15seconds, and at 95° C. for 15 seconds. For the production of standardcurves used for absolute quantification, as standard DNAs, 5-folddilution series of a dsDNA mixed solution of Dap and Thr (31250, 6250,1250, 250, 50, 10, and 0 copy) were used for artificially synthesizedRNA, and 5-fold dilution series of mouse genomic DNA (Clonetech) (31250,6250, 1250, 250, 50, 10, and 0 copy) were used for total RNA. Since cDNAwas ssDNA, the quantitative value was calculated by the formula: adetected value (dsDNA copy)×2. The sequences of individual primers areshown in Table 2. Data analysis was carried out using LightCycler 480Software, Version 1.5 (Roche) or SDS Software 2.1 (Life Technologies).

TABLE 2 Primer sequence (SEQ ID NOS: 17 to 80) Acces- Position sionName of Forward primer sequence Reverse primer from 3′ No. primer(5′→3′) sequence (5′→3′) end (nt) NM_00 Gnb211  TGACCAGAGATGAGACCAACTAAAGTGGGAGTGACCTC 1016 8143 TGG TCAGAGC NM_02 NanogTGATTCTTCTACCAGTCCCAAA TGAGAGAACACAGTCC 1807 8016 CAA GCATCTT NM_01Oct3/4 CCCGGAAGAGAAAGCGAACT CGGGCACTTCAGAAAC  620 3633 ATGG NM_01 Sox2TCTTGCTGGGTTTTGATTCTGC CAAATCCGAATAAACT  543 1443 CCTTCCTTG NM_00 Rex1CTCAAGCCGGGTGCAAGA GCCCGTGGACAAGCAT  893 9556 GT NM_01 Eef1b2CCTTCGCCATGGGATTCG CGCCAGGTAATCGTTG 1619 8796 AGCA NM_00 Atp5a1GTACCCTCCTTCCACCGGG GCCATTGCTGAGGTCA  289 7505 CACAG NM_01 Tubb5CAGTCTGAGACCGGCCCAG TGTGCACGATTTCCCTC 2505 1655 ATG Dap_1856AAATTAGTCATTGCGGGACCG TCGTTCTGCCAATTTAA 1856 CAGCTTC Dap_1795GAACACCACATTTTGACCTTGT GAAAGCATCTGACTCA 1797 AGG ACAGGCA Dap_1653GATTGATTTAACAACGCCCGAA TCCGTGCTCTAATGCAA 1653 TTTTTG Dap_1334GAAAAGCAGCAAGGACATCCG TGCTCCGCTCCTCTTGC 1334 TC Dap_1209GTCAGGCGTTAAACTGTCAGTC TTTATCCCCCCGTCTAA 1209 G TCAATG Dap_962TTTCAATCCGGCCCTTTAGG AGTGCATTGGCAGCGA  962 TCAG Dap_785ACTGCGGAAATTCTTGTGCG CGAAGAAGGTCACGGA  785 ATTCG Dap_686TTTTGGCGCCCACAGTGAT GACAAACTTCGCTATT  686 GTGCCG Dap_539AGCTGAAGCAGCCCGCATA GCGGTCTGGAAGCGTT  539 AGC Dap_367 AATGCGGCTGCACTGGTGCGTCTTTATATTTATGG  367 ATTCCGGC Dap_152 TTCCGTTTCTACTCCTCTGACGAGGCATACTCCACGCCC  152 A G Dap_86 TAAAGAAGCGGGCGTGGAGTA TTTGGATCCTCAGCATC  86 CGTT Thr_2022 CTCGAGATGTGGAAAGGACTTA CATGTAAAGTTAGCGC 2022 TCCCGGTGT Thr_1831  AATGGTTATGGCTGTGGCAAAG CAGCAGCGGAAGTGTT 1831 ACCTGThr_1606 AATTGTCCGTTCCATCTGTGAG GTAAGGGTTGACTGAG 1606 A TTGACAAGGThr_1411 GAAAAACGGCACAGGCCTTC CGATTGCCGCCGCAC 1411 Thr_1065CTGAAAGATCCGAACACAGCG TCAGTCGGCAATGTGA 1065 CAGG Thr_858AGCTGACCGTCTTTGAAAGCG TTCCGGCGACTGTTTCT  858 GTTT Thr_767AGTGGCTAAACGGACCGCA CACCTTCACATGGACA  767 GGAGG Thr_613ATGCTGGCGCTTCTCTCGT TGGGTCTCGTCATCCTC  613 ATG Thr_544CTCGTCGGCGGACTTGTG GGACGCGGATCATTTG  544 GG Thr_439GTGCTGACAAGAGACGCGAGA TTTACGGCATCGGCAT  439 ATGG Thr_295ATGTTCCATCAGCCGTACCG GGCGACGTGCTCTACTT  295 TTGA Thr_200CGGAGCAGGCCCAACG GGGAAATGAAGCGCGA  200 GC

-   Gel Electrophoresis

Each reverse transcription reaction product was diluted with anRNase-free TE buffer (Life Technologies) to result in a volume of 20 μl,and the thus diluted product was subjected to a denaturation treatmentat 70° C. for 10 minutes, and immediately after the treatment, thereaction solution was cooled on ice. Regarding DNA ladder markers, 1μlof E-Gel 1 Kb Plus DNA Ladder (Life Technologies), which was dilutedwith an RNase-free TE buffer to a volume of 20 μl and was then thermallydenatured, was used as an ssDNA ladder, and 1 μl of E-Gel 1 Kb Plus DNALadder (Life Technologies) which was diluted but was not thermallydenatured, was used as a dsDNA ladder. However, in a system of usingMillennium RNA Markers, a thermal denaturation treatment was omittedafter completion of the dilution. The reverse transcription reactionproduct and the ladder marker were filled into 2% E-Gel EX Agarose Gels(Life Technologies), and thereafter, electrophoresis was carried outusing E-Gel iBase Power System (Life Technologies) for 10 minutes.Electrophoretic images were photographed using FAS-Digi (NIPPON GeneticsCo. Ltd, Tokyo, Japan).

Results

-   Selective Cleavage of DNA in RNA-DNA Hybrid by dsDNase and DNase I

First, whether dsDNase or DNase I has an activity of selectivelycleaving DNA in an RNA-DNA hybrid under standard reserve transcriptionbuffer conditions was examined.

Crab-derived double strand-specific nuclease has been known to have anactivity of cleaving DNA in an RNA-DNA hybrid (Non Patent Literature11). However, this enzyme has almost no activity at a temperatureapplied during reverse transcription (Non Patent Literature 11). Assuch, shrimp-derived dsDNase known as a thermally unstable enzyme hasbeen focused (Non Patent Literature 12). This enzyme has an activityaround 37° C. and specifically cleaves double-stranded DNA. Thus, thisenzyme has an action to remove contaminated DNA, genomic DNA and thelike without digesting primers or cDNA (Non Patent Literatures 12 and13). However, it had not yet been reported that this enzyme has anactivity of cleaving DNA in an RNA-DNA hybrid, which is essential forcarrying out a strand displacement reaction. Hence, using Fluorescenceresonance energy transfer (FRET), an analysis was carried out. As aresult, it was found that dsDNase has an activity of selectivelycleaving the DNA in an RNA-DNA hybrid, although the activity is about ahalf of the activity on double-stranded DNA (FIGS. 2A to 2C). Inaddition, it was also confirmed that this enzyme has almost no activityon an RNA strand in an RNA-DNA hybrid, single-stranded DNA, andsingle-stranded RNA (FIGS. 2D to 2F). Similar results were obtained fromheat-labile double-strand specific DNase (HL-dsDNase) having higherthermal instability than the dsDNase, but it was found that the nucleaseactivity of HL-dsDNase is lower than that of dsDNase (FIGS. 2A to 2C).On the other hand, when DNase I as a non-specific DNase was used, itexhibited an activity of selectively cleaving DNA in an RNA-DNA hybrid,as with dsDNase. However, this enzyme also exhibited an activity onsingle-stranded DNA, although the activity was low (FIGS. 2B and 2E). Inview of foregoing, first, an RT-RamDA method of using dsDNase that doesnot exhibit degradation activity on single-stranded DNA was examined.

-   Amplification by Fragmentation of cDNA and Strand Displacement    Reaction

In order to confirm whether dsDNase actually acts on cDNA synthesized bya reverse transcription reaction so as to fragment it, and also, whethera strand displacement reaction thereby takes place and cDNA isamplified, reverse transcription was carried out using about 2 kb ofpoly-A-added artificially synthesized RNA as a template, and thereafter,the presence or absence of fragmentation of cDNA was examined by gelelectrophoresis, and the yield of cDNA was measured by RT-qPCR.Consequently, it was found that, in a standard reverse transcriptionmethod (RamDA (−)), almost no fragmentation took place in both cases ofan oligo dT primer and a random hexamer primer. On the other hand, inRamDA (+), cDNA was fragmented, and a smear electrophoretic image wasobtained (FIG. 3A). It was suggested that these results should beobtained by the action of dsDNase to cleave the cDNA in an RNA-cDNAhybrid. Subsequently, in order to quantitatively detect amplificationcaused by a strand displacement reaction, absolute quantification ofcDNA was carried out by qPCR. As template RNA, artificially synthesizedRNAs, Dap (1,910 b) and Thr (2,052 b), were each mixed in an amount of100 fg each (Dap: 96,725 copies, Thr: 89,991 copies) with 10 pg of totalRNA derived from mouse ES cells (corresponding to a single cell), andthe mixture was then used to carry out a reverse transcription.Quantification was carried out according to qPCR, by designing qPCRprimers in 12 regions from the 3′-end to the 5′-end in each artificiallysynthesized RNA, and measuring the detected amount in each region. As aresult, in both cases of Dap and Thr, an almost constant detected amountwas shown in all of the regions either under the oligo dT primerconditions or under the random hexamer primer conditions, under RamDA(−) control conditions (FIG. 3B). On the other hand, in the case ofRamDA (+), not only under conditions of using the random hexamer primer,but also under conditions of using the oligo dT primer, in regions 500nt or more from the 3′ side, the detected amount of cDNA was stablyincreased to nearly 10 times that of the RamDA (−) control (FIG. 3B).Moreover, it was found that the increased amount was relatively lowaround the 3′-end (FIG. 3B). From these results, it was suggested thatan increase in the amount of cDNA detected in RamDA (+) should beamplification by a strand displacement reaction, and that the nick onthe cDNA side formed by dsDNase would become a starting point of thestrand displacement reaction.

-   dsDNase Enzyme and Reverse Transcriptase that can be Used in    RT-RamDA Method

In order to examine a dsDNase enzyme and reverse transcriptase that canbe used in an RT-RamDA method, whether amplification effects can beobtained even under conditions of using other dsDNase enzymes or varioustypes of reverse transcriptases was examined. First, regarding dsDNaseenzymes, a comparison was made among three enzymes, namely, dsDNase (43KDa) commercially available from ArcticZymes, dsDNase (47 KDa) ofAffymetrix, and duplex specific nuclease (DSN) of Evrogen. Using 10 pgof total RNA derived from mouse ES cells that was the amount of totalRNA corresponding to a single cell, reverse transcription was carriedout. As a result, a significant increase in the detected amount of cDNAwas observed in the two dsDNase-added samples (FIG. 4A). Theamplification rate of DSN was very low because of a difference in activetemperature, but it was found that if the enzyme is a doublestrand-specific DNase, the type of the double strand-specific DNase isnot particularly limited to a specific enzyme.

Next, the presence or absence of amplification effects obtained by theRT-RamDA method was examined using commercially available reversetranscriptase. First, to examine the elongation activity of an enzyme,poly-A-added Millennium RNA Markers (Life Technologies) were used toperform reverse transcription using an oligo dT primer, so that aperformance test was carried out. As a result, it was found that,regardless of a short elongation time that was 2 minutes 30 seconds,almost all enzymes had an elongation activity of 2 kb or more (FIG. 4B).Moreover, reverse transcriptases, which did not have an RNase Hactivity, showed a clear ladder pattern (FIG. 4B, a-c, h, and i),whereas reverse transcriptases having an RNase H activity showed a darksmear pattern (FIG. 4B, e-g and j). When SYBR Gold (Life Technologies)used as a DNA-staining dye is reacted with single-stranded DNA, itprovides fluorescence intensity that is about a half in the case ofreacting with double-stranded DNA (Non Patent Literature 22). Thereby,it is assumed that a difference in the electrophoretic patternsindicates the presence or absence of an RNA-DNA hybrid strand.

Next, individual reverse transcriptases were used to perform reversetranscription using 10 pg of total RNA derived from mouse ES cells,under conditions of addition (+) of RT-RamDA components (dsDNase, a T4gene 32 protein, and a ZNA-random hexamer primer), or non-addition (−)of such components, and a comparison was then made by qPCR, in terms ofthe yield of cDNA. As a result, in all of reverse transcriptases havingno RNase H activity, in the case of RamDA (+), an increase in thedetected amount was observed (FIG. 4C, a-c, h, and i). On the otherhand, in reverse transcriptases having RNase H activity, such anincrease in the detected amount was not observed, or the detected amountwas decreased (FIG. 4C, e-g and j). Moreover, although RNase H activitywas not referred to nominally, AffinityScript also exhibited slightamplification in RamDA (+) (FIG. 4B, d). However, since AffinityScriptshowed a smear pattern in its electrophoretic image, it was assumed thatit had an RNase H activity although the activity was weak (FIG. 4B, d).From these results, it was found that the amplification action of theRT-RamDA method is not limited to specific dsDNase or specific reversetranscriptase, but that this is a widely used technique as long as theenzymes are dsDNases having an activity around 37° C. and reversetranscriptases having no RNase H activity. Furthermore, if an enzyme hadan RNase H activity, it did not show amplification effects. Accordingly,it was demonstrated that strand displacement amplification takes place,directly using RNA as a template.

-   Concerning Method of Enhancing Reaction Efficiency of RT-RamDA    Method

The present inventors have focused on the improvement of anamplification rate, and have developed deviation methods, namely, anRT-RamDA method (RamDA-B) without using a T4 gene 32 protein and anRT-RamDA thermal cycling method (RamDA-C). In a strand displacementreaction, formation of the secondary structure of a template hindersamplification. The present inventors have introduced a T4 gene 32protein into the RT-RamDA method (RamDA-A) as a role for alleviatingsuch a secondary structure. However, at the same time, it was assumedthat the T4 gene 32 protein would cause instabilization of a DNA-RNAhybrid, and would suppress the activity of dsDNase. As such, RamDA-Bwithout using such a T4 gene 32 protein was attempted. Using 10 pg oftotal RNA derived from mouse ES cells as a template, the synthesis ofcDNA was carried out. As a result, it was found that, under theconditions of RamDA-B, the detected amounts of Gnb2l1 and Oct3/4 wereincreased to 40 times or more, in comparison to a standard reversetranscription method (RamDA (−)) (FIG. 5A). On the other hand, regardingNanog, Sox2, Eef1b2 and the like, a great difference was not foundbetween RamDA-B and RamDA-A (FIG. 5A).

Next, RamDA-C, in which only the reaction temperature conditions inRamDA-B were modified and an annealing step and an elongation wererepeated with short intervals, was attempted. As a result, it was foundthat the detected amounts of Gnb2l1 and Oct3/4 in RamDA-C were increasedto 100 times or more those in a standard reverse transcription method(FIG. 5B, RamDA (+) cycle). On the other hand, in the standard reversetranscription method, the effects of thermal cycling were not observed(FIG. 5B, RamDA (−) cycle). In addition, in RamDA-A as well, the effectsof thermal cycling were not observed (the data is not shown). From theseresults, it was suggested that, in the RamDA-B method, the re-annealingof a primer should be extremely effective for amplification of cDNA. Inparticular, the ZNA-random hexamer primer involves addition of twocation units, and the Tm value of the ZNA-random hexamer primer has beendesigned to be approximately 26° C. higher than that of a general randomhexamer primer (Non Patent Literature 14). Thus, the ZNA-random hexamerprimer enables efficient annealing even during a reaction at 37° C., andfurther, it is considered that annealing has been further promoted bythermal cycling.

-   Concerning RT-RamDA Method of Using DNase I

The greatest reason for the use of dsDNase in the RT-RamDA method isthat the dsDNase does not have a nuclease activity on amplified cDNAthat is single-stranded DNA, and a reverse transcription primer.However, it has been known that DNase I that is a non-specific DNAdegrading enzyme originally has a lower nuclease activity onsingle-stranded DNA, than on double-stranded DNA (Non Patent Literature12), and that its nuclease activity is reduced in a monovalent cation(e.g. K+, Na+, etc.) dependent manner (Non Patent Literatures 23 and24). On the other hand, the general composition of a reaction bufferused in reverse transcription (First-Strand buffer (Life Technologies),PrimeScript Buffer (for cDNA synthesis) (TaKaRa), Maxima H Minus FirstStrand cDNA Synthesis RT-Buffer (Thermo Fisher), M-MuLV ReverseTranscriptase Reaction Buffer (New England Biolabs), AffinityScript RTBuffer (Agilent Biotechnology), etc.) comprises 50 mM Tris-HCl, 75 mMKCl, and 3 mM MgCl₂. Thus, the concentrations of Tris-HCl and KCl arehigh in such a reaction buffer used in reverse transcription. That is,it was predicted that the nuclease activity of DNase I onsingle-stranded DNA would be low under the conditions of the reversetranscription reaction buffer, and the RT-RamDA method would function.In reality, according to FRET analysis, nuclease activity was measuredon the basis of 20 mM Tris-HCl, 50 mM KCl and 2 mM MgCl comprised inDNase I reaction buffer (Life Technologies), by increasing theconcentrations of Tris-HCl and KCl. As a result, it was found that thenuclease activity of DNase I, not only on double-stranded DNA, but alsoon single-stranded DNA and DNA in an RNA-DNA hybrid, is significantlyinhibited under conditions of high concentrations of Tris-HCl and KCl(FIG. 6). In the concentrations of Tris-HCl and KCl that were equivalentto those in a reverse transcription reaction buffer, the activity wassuppressed to 43%, 16%, and 14%, in comparison to the conditions of aDNase I reaction buffer (FIG. 6, ##).

Next, the efficacy of the RT-RamDA method of using a non-specific DNAdegrading enzyme, and in particular, the relationship between the saltconcentration in the reaction solution and an amplification rate wasexamined. The salt concentration was changed from the composition of aDNase I reaction buffer to the composition of a reverse transcriptionreaction buffer, and whether the RT-RamDA method functions was examinedusing qPCR. As a result, unexpectedly, it was found that the RT-RamDAmethod provides sufficient amplification even under conditions of aDNase I reaction buffer having a high nuclease activity onsingle-stranded DNA (FIG. 7). Moreover, even if the concentrations ofKCl and Tris-HCl were changed, it had only a small influence on theamplification rate (FIG. 7, a-i). On the other hand, it was found thatthe concentration of NaCl in the composition of a reverse transcriptionreaction buffer has a great influence on the amplification rate (FIG. 7,j-l). From these results, it is assumed that the composition of areaction buffer containing no NaCl is desirable. Moreover, even in thecase of using an in-house reaction buffer, the in-house reaction bufferexhibited an amplification rate that was not inferior to commerciallyavailable First-Strand buffer (Life Technologies) or PrimeScript Buffer(for Real Time) (TaKaRa) (FIG. 7, j, FS, and PS). From these results, itwas found that the reaction buffer used in the RT-RamDA method is notlimited to a certain reaction buffer, and further that the RT-RamDAmethod functions non-dependently on a salt-concentration-dependentnuclease activity. From these findings, it was suggested that factorsother than the salt concentration in the reaction buffer should suppressthe decomposition of cDNA.

-   Contribution of T4 Gene 32 Protein to Protection of cDNA and    Stabilization of Amplification

It has been reported that the T4 gene 32 protein has an action to bindto single-stranded DNA, so as to protect the single-stranded DNA fromnuclease (Non Patent Literature 25). Thus, the fragmentation state ofcDNA according to the RT-RamDA method of using, as a template,poly-A-added artificially synthesized RNA, and also using only an oligodT primer, was examined based on the presence or absence of a T4 gene 32protein (FIG. 8). As a result, it was found from the electrophoreticpatterns that, in both cases of DNase I and dsDNase, the fragmentationof cDNA is suppressed by combining the enzyme with a T4 gene 32 protein,rather than in the case of using DNase alone (FIG. 8, A). In theanalysis using BioAnalyzer, not only similar results could be obtained,but it was also found that the yield of cDNA was increased to nearly 10times by allowing DNase to act thereon (FIG. 8, B). It is consideredthat these results suggest global amplification of cDNA by a stranddisplacement reaction. Subsequently, using qPCR, the amplification ratefrom the 3′-end to the 5′-end in template RNA was examined (FIG. 9). Asa result, in the case of using DNase I alone, almost no amplificationwas observed. In contrast, when DNase I was combined with a T4 gene 32protein, amplification was observed at a magnification of nearly 10times (FIGS. 9A and 9B). Furthermore, it was found that theamplification rate on the 3′ side was improved in comparison to dsDNase(FIG. 9,A). On the other hand, in the case of dsDNase, since dsDNasedoes not originally have a degradation activity on single-stranded DNA,it exhibited a high amplification rate, even when it was allowed to actalone. However, dsDNase resulted in a large fluctuation in theamplification rates on single RNA (FIGS. 9A and 9C). Since theseconditions were almost equivalent to the conditions of RamDA-B, it wassuggested that a fluctuation in the amplification, not only among genes,but also within a single gene, should be increased, unless there is a T4gene 32 protein. From these results, it was suggested that, in RamDA-Dof using DNase I, the T4 gene 32 protein should have two roles forcontributing suppression of the decomposition of cDNA and stabilizationof the amplification rate among genes or within a single gene.

-   T4 Gene 32 Protein Having Action to Improve Ratio Between Nuclease    Activity on DNA in RNA-DNA Hybrid Strand and Activity on    Single-stranded DNA

For the function of the RT-RamDA method of using DNase I, it isconsidered important that the decomposed amount of amplified DNA issmaller than the amount of strand displacement amplification caused byformation of a nick in an RNA-cDNA strand, namely, that nucleaseactivity on single-stranded DNA is sufficiently smaller than activity onDNA in an RNA-DNA hybrid strand. From an experiment regardingfragmentation of cDNA and amplification rate (FIGS. 8 and 9), the T4gene 32 protein was assumed to contribute thereto. Hence, according toFRET analysis, the action of the T4 gene 32 protein on nuclease activityin a reverse transcription reaction buffer was examined (FIG. 10). As aresult, it was found that the activity on the single-stranded DNA wasreduced to 25% by adding the T4 gene 32 protein into the reversetranscription reaction buffer. In contrast, there was almost nofluctuation in the activity on double-stranded DNA, and the activity onDNA in the RNA-DNA hybrid strand was approximately 60% (FIG. 10, A).Subsequently, the ratio of the activity on single-stranded DNA to thenuclease activity on DNA in an RNA-DNA hybrid strand was examined. As aresult, it was found that the activity ratio was improved from 40% to14% by addition of the T4 gene 32 protein (FIG. 10, B). This improvementof the ratio is assumed to be a key factor for the efficacy of theRT-RamDA method. On the other hand, in the case of dsDNase, the activityratio was merely approximately 7% even in the absence of the T4 gene 32protein, and thus, it is considered that RamDA-B and RamDA-C withoutusing T4 gene 32 proteins sufficiently function (FIG. 10, B).

-   RamDA-D Showing Reaction Time-dependent Amplification Rate

Whether the amplification rate would be improved in a reactiontime-dependent manner was examined using a 1-cell lysate of mouse EScells as a template (FIG. 11). A relative value of the yield of cDNA wasmeasured by qPCR. As a result, it was found that as the reaction time at37° C. was increased to 30, 60, and 120 minutes, the yield was alsoincreased to approximately 10, 20, and 30 times, respectively (FIG. 11).Such a phenomenon was not observed in RamDA-A of using dsDNase (the datais not shown). The nuclease activity of dsDNase was significantlyinhibited in a reverse transcription reaction buffer, in particular, inthe presence of a T4 gene 32 protein, and the nuclease activity ofdsDNase on DNA in an RNA-DNA hybrid was only about 6%, in comparison tothe nuclease activity of DNase I (FIG. 10, A). This may cause adifference in the amplification rate between the use of DNase I and theuse of dsDNase. On the other hand, it was confirmed that RamDA-Dfunctions without problems even in a crude sample containing impurities,such as a cell lysate. Moreover, with regard to the nuclease activity ondouble-stranded DNA in a reverse transcription reaction, the activity ofDNase I was higher than the activity of dsDNase, and thus, it wassuggested that RamDA-D should be effective regarding not only anamplification rate, but also an ability to remove contamination (FIG.10, A).

-   RamDA-D that is Not Limited to Specific RNase H Minus Reverse    Transcriptase, and in Addition, Functions Also on RNA Corresponding    to 100 Cells.

In order to confirm whether RamDA-D is limited to a specific RNase Hminus reverse transcriptase, an examination was carried out usingreverse transcriptase of SuperScript series (Life Technologies), as wellas PrimeScript RT Enzyme Mix I (FIG. 12). As a result, amplification ofcDNA could be confirmed in SuperScript II and III. Among others,SuperScript II exhibited an amplification rate that was equivalent tothat of PrimeScript RT Enzyme Mix I (FIG. 12, SSII). Furthermore, it wasconfirmed that RamDA-D functions even in the case of using Maxima HMinus First Strand cDNA Synthesis Kit (Thermo Fisher) or ReverTra AceqPCR RT Kit (TOYOBO) (the data is not shown). From these results, it wasfound that RamDA-D is not limited to a specific RNase H minus reversetranscriptase. Further, it was also found that RamDA-D functions withoutproblems even in RNA in an amount greater than 10 pg of total RNAcorresponding to a single cell, for example, in 200 pg of RNA or 1 ng ofRNA (FIG. 12, 200 pg, 1 ng). From these results, it was suggested thatsufficient amplification performance should be ensured even usingtemplate RNA in an amount of at least 100 cells.

As stated above, the RT-RamDA method can be an extremely useful means asa technique of amplifying trace RNA, and in particular, theamplification rate provided by RamDA-C should be an extremely greatadvantage in the analysis of detecting a specific target gene. On theother hand, since RamDA-C causes a large fluctuation in theamplification rates among genes, RamDA-A involving the use of a T4 gene32 protein is effective for an analysis requiring uniform amplification.Moreover, RamDA-D involving the use of such a T4 gene 32 protein andDNase I as a non-specific DNA degrading enzyme has a high amplificationrate and also has a small amplification fluctuation among genes orwithin a single gene. Furthermore, since the nuclease activity of DNaseI on double-stranded DNA during a reverse transcription reaction is alsomaintained at a level higher than that of a double strand-specific DNAdegrading enzyme, it is expected that RamDA-D will have the effect ofreducing the influence of DNA contamination. As described above, RamDA-Dis a method comprising both the superiority of RamDA-A and that ofRamDA-B, and this is an extremely effective and simple reversetranscription method targeting a trace amount of RNA.

The present application claims priority from Japanese Patent ApplicationNo. 2014-200258 (filed on Sep. 30, 2014); the disclosure of which ishereby incorporated by reference in its entirety. In addition, allpatent publications, patent applications, and publications cited hereinare incorporated herein by reference in their entirety.

1. A kit, comprising: a degrading enzyme specific to DNA in an RNA-DNAhybrid, and an RNase H minus reverse transcriptase, wherein: thedegrading enzyme specific to DNA in an RNA-DNA hybrid is a doublestrand-specific DNA degrading enzyme or a non-specific DNA degradingenzyme, and if the degrading enzyme specific to DNA in an RNA-DNA hybridis a non-specific DNA degrading enzyme, then the kit further comprises asingle-stranded DNA-binding protein.
 2. The kit according to claim 1,wherein the double strand-specific DNA degrading enzyme is aCrustacea-derived double strand-specific DNA degrading enzyme or avariant thereof.
 3. The kit according to claim 1 wherein the doublestrand-specific DNA degrading enzyme is selected from the groupconsisting of Solenocera melantho (coastal mud shrimp) DNase, Penaeusjaponicus (prawn) DNase, Paralithodes camtschaticus (king crab) DSN,Pandalus borealis (Northern shrimp) dsDNase, Chionoecetes opilio (snowcrab) DSN, and other DSN homologs.
 4. The kit according to claim 1,wherein the single-stranded DNA-binding protein is T4 gene 32 protein.5. The kit according to claim 1, further comprising a substrate.
 6. Thekit according to claim 1, further comprising a primer.
 7. The kitaccording to claim 5, further comprising a primer.